Fragment-Resolved Carbon and Hydrogen Isotope Analysis of Urinary Steroids by Orbitrap Mass Spectrometry

Compound-specific stable isotope analysis (CSIA) is widely used to distinguish sources and transformation pathways of organic compounds. In steroid CSIA each analyte is represented by a single molecular-average isotope value, which can limit discrimination when sources overlap in isotopic composition. This limitation is especially important in anti-doping analysis, where molecular-average δ13C measurements can be inconclusive for compounds whose C-isotope composition falls within the endogenous range. Here, we present an Orbitrap-based method for fragment-resolved intramolecular isotope analysis of steroids. Using urinary androsterone as a model analyte, we measured δ13C values for four diagnostic ions and δD for one fragment following controlled administration of androstenedione and testosterone undecanoate. The resulting multidimensional isotope fingerprints reveal responses not evident from bulk δ13C measurements alone, including cases in which administered androgen lies near the endogenous δ13C range. Time-dependent differences among fragment ions further suggest sensitivity to changes in metabolic routing and pool mixing during steroid transformation. These results establish fragment-resolved isotope analysis by Orbitrap mass spectrometry as a promising extension of conventional steroid CSIA and a potential new tool for forensic steroid source discrimination, with possible applications to environmental steroids and geological sterols and steranes.